1. Field of the Invention
The present invention is in the field of chemical analysis usually performed along with physical separation of analytes from solution by means of a solid phase. If there are multiple analytes some remain in the solution phase and others may attach to the solid phase. The attachment is said to occur by means of ligand pairs, where one of the pair of ligands is permanently attached to the solid phase and the other of the ligand pair is a characteristic of at least one of the analytes to be detected. The separation process permits separate identification and detection of multiple analytes when the characteristic of the ligand is present on one but not all of the analyte molecule species. Common application of such phase separations is practiced in Enzyme Ligand Immuno Assay (ELISA) and in chromatography. By contrast, two phase optical assays are designed to overcome the common steps of physically separating the analytes for analysis in separate compartments by applying optical analysis to both the solid phase and the solution phase in one container.
The present invention is thus broadly concerned with improved two-phase assays normally carried out in a single container or cuvette using an assay mixture including a buffer, a plurality of particles or beads, an affinity (ligand) agent, a sample to be assayed, and a marker. More particularly, it is concerned with such assays wherein settling and phase separation of the assay mixture after incubation may be monitored to a steady state condition, whereupon assay data can be generated using a source of electromagnetic radiation in a dual sensor or single sensor system. The invention also takes advantage of timely measurements of optical phases during the settling process. The assays of the invention may also be used to identify and/or quantitate multiple analytes in a single sample.
2. Description of the Prior Art
U.S. Pat. No. 5,674,699 (incorporated by reference herein) describes the use of a suspension of affinity-specific micro beads in order to adsorb one specific ligand species out of a series that may be present in a given sample to be assayed. However, instead of eluting the specific ligand as a separate fraction, as is common in conventional chromatography, the measurement of the adhered ligand is performed while attached to the micro beads, in a special container. In order to perform these measurements, the '699 patent provides a container configured so that some of the micro beads and suspending liquid are transferred to a capillary, stoppered, and centrifuged. The capillary forms the measurement container.
Two methods are described for measuring the adhered ligand. One is by absorbance of light of a specific wavelength by the ligand. Since the micro beads are transparent, light is passed through them and only light that is absorbed by the analyte is attenuated. A second wavelength, not absorbed by the analyte, is used as an optical correction for the roundness of the capillary container and for the slight light scatter from the micro beads. A second analyte, present in the sample, but not having the specific ligand characteristic remains in the solution phase and is measured separately in the said solution phase.
The other method of measurement is fluorescence quenching, wherein a fluorescent dye which absorbs light at the same wavelength as the analytes is dissolved in the suspending liquid. Dye solution surrounds the micro beads and penetrates the micro beads to the same extent as the analyte itself. In the presence of analyte, there is less fluorescence because the light is absorbed by the analyte and is not available for excitation of the fluorescent dye. This second method is less dependent upon the complete transparency of the micro beads, but is nevertheless linearly related to the amount of analyte present both in the supernatant, solution phase, and in the micro bead lower phase, after the micro beads settle.
The apparatus and methods described in the '699 patent are specialized and somewhat cumbersome and time-consuming to use. That is, the container apparatus having the separate mixing and measuring sub-compartments, and the need for centrifugation, are limits upon the commercial utility of the assays described in this patent.